The objective of the proposed studies is to continue studies on understanding the biochemical mechanism(s) responsible for maintaining the clarity of the lens and those that lead to the formation of senile and diabetic cataracts. A reliable, reproducible, and sensitive digital image analysis method will be developed to quantitate the alterations in transmitted light during cataractogenesis. A fluorometric variant of this technique will be used for the determination of lens constituents such as free intracellular calcium, and sodium, lipid peroxides and also for the measurement of intracellular pH and transmembrane voltage. In addition, a single lens fiber cell model for studying cataractogenesis will also be developed. The proposed studies are aimed at understanding the contribution of polyol pathway, nonenzymatic glycosylation and perturbed redox state to cataractogenesis induced by hyperglycemia and oxidative stress generated by free radicals and sulfhydryl oxidation. The role of free intracellular calcium, increased proteolysis and changes in the redox state of the lens leading to alterations in NAD(P)H/NAD(P) and GSH/GSSG ratio will be investigated. The mechanism of activation of lens aldose reductase by glycosylation and oxidation will also be studied and the amino acid residues modified will be identified. Furthermore, the proteolytic susceptibility of crystallins modified by oxidative stress and/or nonenzymatic glycosylation will be investigated. Studies on the mechanism of transport of electrophilic xenobiotics and oxidized glutathione and the interrelationship between the two transport systems will demonstrate the role of the transport of oxidized glutathione and glutathione-xenobiotic in determining the redox state of the lens, and show how electrophilic xenobiotics affect GSSG levels and how increased oxidative stress affects detoxification of xenobiotics. These studies will identify the role of oxidative stress and non-enzymatic glycosylation in protein modification, proteolysis, and crystallin aggregation and will quantitatively elucidate the mechanisms by which intracellular ionic changes contribute to cataractogenesis.